PROJECT SUMMARY/ABSTRACT My overarching goal is to understand the three-dimensional (3D) enhancer regulation of aging and age-related diseases. Aging is accompanied by functional decline of tissues and an increased probability of many diseases. Epigenetic alteration is one hallmark of aging. Several lines of evidence demonstrate that histone acetylation levels elevate in senescent cells and global remodeling of the enhancer landscape occurs in aging and senescence. However, there is poor understanding of the influence of three-dimensional (3D) enhancer regulation changes during senescence. My preliminary data demonstrate that many enhancers connect together to form large clusters, termed hubs. The enhancers and promoters included in hubs are different between proliferation and senescence. The senescence-specific hubs ensure critical transcription programs for senescent phenotypes. However, how enhancer hubs change in response to stress and the mechanisms and functions of these alterations remain to be elucidated. The main goal of the proposed studies is to uncover mechanisms underlying the 3D regulation of enhancer hubs under different sources of stress during aging and age-related disease. I hypothesize that (1) during aging, key enhancers cluster together within hubs to regulate stress-specific gene expression programs; (2) two enhancer regulatory pathways exist during normal aging: one is cell intrinsic, and the other is cell extrinsic deriving from environmental inflammatory signals which accelerate the intrinsic pathway. In the mentored K99 phase (Aim 1), I will study epigenetic mechanisms regulating 3D enhancer hubs in senescence (Aim 1a) and test their generalization among different types of senescence (Aim 1b). Completion of these aims will set the stage for my independent research in aging and age-related metabolic disorder. In the R00 phase (Aim 2), in natural aging in liver, utilizing a unique mouse model, I will dissect the intrinsic enhancer regulatory pathway in hepatocytes and the extrinsic pathway stimulated by environmental inflammatory signals. This will provide potential targets to ameliorate age-related metabolic disorders in future direction of my independent lab. Ultimately, the proposed research will unveil new mechanisms underlying the interplay between inflammation and enhancer regulation in aging and provide novel therapeutic targets for age-related diseases. The career development and training components in K99 phase will expand my experience in aging, metabolism and immunology to provide a rich foundation for my successful transition to an independent career.